PCR/qPCR reaction set-up with mosquito low-volume liquid handlers

TTP Labtech’s mosquito range of automated pipetting systems are ideal for the handling of large sample numbers in PCR and qPCR set up, reducing manual set-up time and the potential for errors. PCR and qPCR reaction set-up requires considerable pipetting skills as even very small pipetting errors results in significant variations in the end results.

positive displacement technology, essential in accurate and precise pipetting of small volumes and resulting in robust data

disposable tips eliminate cross-contamination

easy to use software provides fast reaction setup for low- to high-throughput applications

capable of full integration with other liquid handlers and ancillary equipment

case study 1: low-cost SYBR-Green qPCR reaction set-up

This study at Dr. Mark Watson’s Lab (Murdoch University, Perth, Australia), performed by John Blinco, uses mosquito HV to miniaturise the reaction volumes in a novel qPCR gene expression (GEX) study. 11 human immune cell lines were simulated with up to 6 different peptides. 6 different genes (including 2 housekeeping genes) were studied via GEX analysis using FastStart Universal SYBR Green kit (Roche Diagnostics, USA). The qPCR reactions were prepared using 1 μL cDNA in a total volume of 5 μL and in 384-well plates.

The figure shows the amplification results of 56 samples for 6 genes of interest. Using mosquito HV did not affect the amplification profile of these genes. By miniaturising the reaction volumes a 4-fold reduction in reaction volume, sample input and cost was achieved. More importantly, the lower reaction volume allows valuable and limited samples to be analysed multiple times from the same up-stream reaction (for example, a patient-derived or archival sample).

case study 2: low-cost KAPA library quantification sample prep

qPCR-based quantification kits (Kapa Biosystems, Wilmington, USA) are commonly used to accurately quantify cDNA or NGS libraries. In this study, utilising precise and accurate automated liquid handling technology of mosquito HV the qPCR reaction volumes are accurately miniaturised, resulting in significant cost savings during the quantification process. 6 different DNA libraries at an average size of 490 bp were prepared using TruSeq Nano DNA library prep kit (Illumina Inc., USA). The libraries were quantified using KAPA library quantification kit (code: KK4824) at a total volume of 10 μL in triplicate, both manually and using mosquito HV. This is a two-fold reduction in volume compared to standard reactions.

The figure compares the use of a manual system and the mosquito HV for determining concentrations of DNA libraries. The percentage of errors for each methods are shown.